NMDA receptor (Kd = 1.4 μM)

d -对映体是NMDA谷氨酸受体（受体，n -甲基- d -天冬氨酸）的有效特异性拮抗剂。L型对NMDA受体无活性，但可能影响AP4(2-氨基-4-磷酸丁酸酯；兴奋性氨基酸受体。

D-AP5是一种NMDA 受体拮抗剂。长期脑室内 D-AP5 输注会以平行的剂量依赖性方式对体内的长时程增强 (LTP) 和空间学习产生不利影响。当空间学习被阻止时，D-AP5 的大脑浓度不会导致任何可检测到的感觉障碍 [2]。在试验期间，游泳速度的逐渐降低与 D-AP5 的输注有关。当受 D-AP5 影响的动物无法学习时，它们在空间任务中会出现感觉运动异常，并且随着时间的推移会变得更糟。水迷宫的延迟匹配位置方案揭示了用 D-AP5 治疗的大鼠存在延迟依赖性空间记忆缺陷 [3]。

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本系列实验研究了NMDA受体拮抗剂d -2-氨基-5-磷酸戊酸酯（D-AP5）是否能在与体内海马长期增强（LTP）损伤相当的剂量范围内诱导空间学习损伤。利用微透析技术估计海马D-AP5的细胞外浓度，以比较这些损伤是否发生在与体外海马切片中损害LTP所需的浓度相似的浓度。将D-AP5以一定浓度（0 ~ 50 mM）通过渗透微型泵长期注入大鼠侧脑室。他们首先接受的训练是在一个开阔的水迷宫任务中找到并逃到一个隐藏的平台上。行为学习结束后，他们被聚氨酯麻醉，并试图唤醒和监测海马LTP。采用微透析法提取海马D-AP5细胞外样本，最后处死动物，解剖组织样本。采用荧光高效液相色谱法分析微透析和组织样品中D-AP5的含量。结果表明，首先，D-AP5对空间学习的损害呈剂量线性依赖关系，与体内海马LTP的损害高度相关。没有观察到D-AP5浓度在不影响学习的情况下阻断LTP。其次，微透析估计表明，在某些假设下，D-AP5在细胞外浓度下导致这些损伤，与体外损害LTP的浓度相当。第三，全组织和微透析样品的比较显示，浓度比约为30:1，表明97%的脑内D-AP5是透析探针无法接近的。研究发现，输注20 mM EGTA可导致透析灌注液中D-AP5增加7倍，这表明至少部分无法进入的D-AP5被钙依赖机制捕获。进一步的两项行为控制研究表明，D-AP5诱导的空间学习障碍不太可能继发于药物诱导的运动障碍，并且D-AP5浓度刚好足以完全阻断海马LTP的组的表现与伊博滕酸诱导的双侧海马病变组的表现在统计学上没有区别。综上所述，这些发现为NMDA受体的激活对于某些类型的学习是必要的这一假设提供了支持。[2]

Drug concentrations, surgery, amino acid analysis, and histology All drugs and anesthetics were made up in deionized water except in a final replicate when they were made up in pyrogen-free water. An equivalent stock concentration of 100 mM D-AP5 was made up in 100 mM NaOH and kept as frozen aliquots. This was diluted with aCSF for a range of concentrations (5, 13, 20, 30, 40, and 50 mM) “spiked” with NaOH to maintain a pH of 7.4. Stock solution of EGTA (500 mM) was made up as an equivalent in NaOH and diluted to 20 mM with Ca*+-free aCSF. Ibotenic acid was dissolved in phosphate-buffered saline (pH 7.4) at a concentration of 10 mg/ml. As D-AP5 does not cross the blood-brain barrier, it was delivered to the brain by chronic infusion using osmotic minipumps (Alza model 2002; pumping rate, 0.5 pl/hr). Animals were placed in a Kopf stereotaxic apparatus to implant the minipumps under tribromoethanol anesthesia (0.29 gm/kg). An incision was made along the midline, the skull was exposed, and an L-shaped stainless steel cannula (23 gauge) was placed in the left lateral ventricle (AP = -0.9, ML = 1.3, DV = -4.5 from skull surface). The cannula was attached to the minipump with Silastic tubing and secured in place with dental acrylic and three watchmaker’s screws. The minipump was placed in the subcutaneous pocket extending from the caudal end of the incision to the shoulder blades. The scalp incision was closed with discontinuous suture, and animals were allowed 2 d postoperative recovery. During the surgery, positions were marked on the skull for the microdialysis probe and the two electrodes. For the ibotenic acid lesions, animals were placed in a stereotaxic frame and anesthetized with tribromoethanol. Animals were lesioned according to the methodology devised by Jarrard (1989) using ibotenic acid (either 0.05 or 0.10 ~1) at 26 sites bilaterally and allowed 2 weeks recovery before behavioral testing. Analysis of D-AP5 and amino acids in both brain tissue and dialysis samples was carried out using a Varian Vista 5500 pumping system 9090 automatic column injector, and a 5 pm Nucleosil C-18 column (250 x 4.6 mm). Separation ofamino acids was achieved with a gradient eluent using a phosphate buffer [buffer A: 50 mM and tetrahyarofuran (THF) (2.5%); pH 5.121 with an organic modifier, methanol [buffer B and THF (1.25%)]. The gradient profile with a pumping rate of 1 ml/ min was as follows: [time (min), %B] 0, 0; 5, 0; 7, 25; 15, 50; 23, 60; 25.90: 28. 110: 32.100: 42.0. Precolumn derivatization with o-nhthaldehydk (L&d&h et al.,’ 19i5) allowed detection of primary amino acids by fluorescence, using an ABS 980 fluorescence detector (excitation wavelength, 230 pm; emission wavelength, 1398 pm). A standard containing known quantities of amino acids and D-AP5 was injected onto the column at the start and end of each daily session of analysis to calibrate retention time and peak area of each molecular component measured. Data were integrated and quantified using a microcomputer-based integration package. The animals were killed at the end of the experiment, and their brains were removed on ice. Tissue from the right and left hippocampus of animals in the dose-response study was dissected out and kept in frozen storage (-20°C) for analysis of D-AP5 and amino acid content. Tissue from the region immediately adjacent to the infusion cannula was retained in formalin, frozen, cut into 30 pm coronal sections, and stained with fast cresyl violet. This allowed verification of the cannula position and assessment of any damage caused by it and/or by drug infusion. The brains from the hippocampally lesioned animals were embedded in egg yolk, frozen, and cut into 30 pm horizontal sections to assess the extent of cell loss.[2]

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Three experiments were conducted to contrast the hypothesis that hippocampal N-methyl-d-aspartate (NMDA) receptors participate directly in the mechanisms of hippocampus-dependent learning with an alternative view that apparent impairments of learning induced by NMDA receptor antagonists arise because of drug-induced neuropathological and/or sensorimotor disturbances. In experiment 1, rats given a chronic i.c.v. infusion of d-AP5 (30 mm) at 0.5 μL/h were selectively impaired, relative to aCSF-infused animals, in place but not cued navigation learning when they were trained during the 14-day drug infusion period, but were unimpaired on both tasks if trained 11 days after the minipumps were exhausted. d-AP5 caused sensorimotor disturbances in the spatial task, but these gradually worsened as the animals failed to learn. Histological assessment of potential neuropathological changes revealed no abnormalities in d-AP5-treated rats whether killed during or after chronic drug infusion. In experiment 2, a deficit in spatial learning was also apparent in d-AP5-treated rats trained on a spatial reference memory task involving two identical but visible platforms, a task chosen and shown to minimise sensorimotor disturbances. HPLC was used to identify the presence of d-AP5 in selected brain areas. In Experiment 3, rats treated with d-AP5 showed a delay-dependent deficit in spatial memory in the delayed matching-to-place protocol for the water maze. These data are discussed with respect to the learning mechanism and sensorimotor accounts of the impact of NMDA receptor antagonists on brain function. We argue that NMDA receptor mechanisms participate directly in spatial learning.[3]

[1]. The effects of a series of omega-phosphonic alpha-carboxylic amino acids on electrically evoked and excitant amino acid-induced responses in isolated spinal cord preparations. Br J Pharmacol. 1982 Jan;75(1):65-75.

[2]. The NMDA receptor antagonist D-2-amino-5-phosphonopentanoate (D-AP5) impairs spatial learning and LTP in vivo at intracerebral concentrations comparable to those that block LTP in vitro. J Neurosci. 1992 Jan;12(1):21-34.

[3]. N-methyl-d-aspartate receptors, learning and memory: chronic intraventricular infusion of the NMDA receptor antagonist d-AP5 interacts directly with the neural mechanisms of spatial learning. Eur J Neurosci. 2013 Mar;37(5):700-17.

The D-enantiomer is a potent and specific antagonist of NMDA glutamate receptors (RECEPTORS, N-METHYL-D-ASPARTATE). The L form is inactive at NMDA receptors but may affect the AP4 (2-amino-4-phosphonobutyrate; APB) excitatory amino acid receptors.

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1 The depressant actions on evoked electrical activity and the excitant amino acid antagonist properties of a range of omega-phosphonic alpha-carboxylic amino acids have been investigated in the isolated spinal cord preparations of the frog or immature rat. 2 When tested on dorsal root-evoked ventral root potentials, members of the homologous series from 2- amino-5-phosphonovaleric acid to 2-amino-8-phosphonooctanoic acid showed depressant actions which correlated with the ability of the substances to antagonize selectivity motoneuronal depolarizations induced by N-methyl-D-aspartate. 3 2-Amino-5-phosphonovalerate was the most potent substance of the series giving an apparent KD of 1.4 microM for the antagonism of responses to N-methyl-D-aspartate. 4 A comparison of the (+)- and (-)-forms of 2-amino-5-phosphonovalerate indicated that the N-methyl-D-aspartate antagonist activity and the neuronal depressant action of this substance were both due mainly to the (-)-isomer. 5 The (-)- and (+)-forms of 2-amino-4-phosphonobutyrate had different actions. The (-)-forms of this substance had a relatively weak and non-selective antagonist action on depolarizations induced by N-methyl-D-aspartate, quisqualate and kainate and a similarly weak depressant effect when tested on evoked electrical activity. The (+)-form was more potent than he (-)-form in depressing electrically evoked activity but did not antagonize responses to amino acid excitants. At concentrations higher than those required to depress electrically evoked activity, the (+)-form produced depolarization. This action was blocked by 2-amino-5-phosphonovalerate.[1]

C5H10NO5P-2

195.1104

197.045

C, 30.47; H, 6.14; N, 7.11; O, 40.58; P, 15.71

79055-68-8

DL-AP5;76326-31-3;L-AP5;79055-67-7

135342

White to off-white solid powder

1.5±0.1 g/cm3

482.1±55.0 °C at 760 mmHg

245.4±31.5 °C

0.0±2.6 mmHg at 25°C

1.536

-2.32

130.66

4

6

5

12

200

1

C(C[C@H](C(=O)O)N)CP(=O)(O)O

VOROEQBFPPIACJ-SCSAIBSYSA-N

InChI=1S/C5H12NO5P/c6-4(5(7)8)2-1-3-12(9,10)11/h4H,1-3,6H2,(H,7,8)(H2,9,10,11)/t4-/m1/s1

(2R)-2-amino-5-phosphonopentanoic acid

D-AP5; (R)-2-Amino-5-phosphonopentanoic acid; 5-Phosphono-D-norvaline; (2R)-2-amino-5-phosphonopentanoic acid; d-APV; D-Norvaline, 5-phosphono-; D-(-)-2-Amino-5-phosphonopentanoic Acid;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~27.78 mg/mL (~140.92 mM)
DMSO :< 1 mg/mL
Ethanol :< 1 mg/mL

配方 1 中的溶解度: 100 mg/mL (507.28 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。